The mammalian cellular response to genotoxic damage is often analyzed using a battery of tests, which often include in vitro chromosomal aberration or micronuclei formation tests. Both of these tests visualize DNA damage in cells after exposure to potential genotoxicants by analyzing harvested chromosomes for aberrations (S. M. Galloway, Environ Mol Mutagen (2000) 35:191-201) or by examining micronuclei formed in cells whose DNA has been damaged (W. von der Hude et al., Mutation Res (2000) 468:137-63). However, there are significant problems with interpreting the results of the currently used in vitro genotoxic tests. False positive results in these tests are not uncommon, and the subsequent analysis, which involves in vivo animal testing, can be costly and time consuming. Assays which can better predict the genotoxic potential of a compound are needed (see, e.g., R. K. Newton et al., Environ Health Persp (2004) 112:420-22). In response to genotoxic stress, cycling cells that are undergoing cellular differentiation are arrested at discrete stages in the cell cycle (see, e.g., T. Weinert et al., Nature Gen (1999) 21:151-52; T. Weinert, Cell (1998) 94:555-58). This differentiation arrest is thought to result from activation of key regulatory kinases and other components in response to DNA damage at critical checkpoints in the cell cycle (T. Weinert (1998) supra; B. S. Zhou-Bin et al., Nature Rev Cancer (2004) 4:216-25).
P. L. Puri et al., Nature Gen (2002) 32:585-93 investigated the ability of four known genotoxic agents (methyl-methane sulfonate, cisplatin, etoposide, and ionizing radiation) to inhibit the differentiation of C2C12 myoblast cells into myotubes. Effects of the agents were also examined by assaying the expression of muscle-specific proteins (myogenin, myosin heavy chain, MyoD), and using a luciferase reporter gene coupled to the muscle creatinine kinase promoter.
The murine gene DDA3 was sequenced for study due to its regulation by p53 (P.-K. Lo et al., Oncogene (1999) 18:7765-74). P-K Lo et al. also found that DDA3 was upregulated in NIH3T3 cells exposed to DNA damaging agents such as adriamycin and mitomycin C. P-K Lo et al. found that DDA3 was strongly expressed in brain, spleen, and lung (with moderate expression in kidney): no expression or minimal expression was found in heart, liver, skeletal muscle, or testis. The 5′ genomic sequence (including the upstream regulatory region) was sequenced and described by S.-C. Hsieh et al., Oncogene (2002) 21:3050-57, who identified the p53-binding element and determined that expression was also induced by p73. P.-K. Lo & F.-F. Wang, Biochim Biophys Acta (2002) 1579:214-18 reported the identification and sequencing of the human DDA3 homolog, also finding that it was expressed in nearly every tissue except adult skeletal muscle. P.-K. Lo & F.-F. Wang, Arch Biochem Biophys (2004) 425:221-32 reported that murine DDA3 is transcribed or edited into a number of different forms.
Tugendreich et al., WO2004/037200, disclosed the measurement of genomic responses of rat liver cells to hydroxyurea, cytarabine, doxorubicin, ifosfamide, thioguanine, azathioprine, etoposide, and albendazole, each administered in vivo. The genomic responses were then used to derive a “drug signature” that correlates the transcriptional regulation of two genes (aminolevulinate synthase 2 delta, Genbank NM 013197; and peripherin 1, Genbank NM 012633) with the propensity of each compound to cause depletion of reticulocytes.